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Partial Restoration of Antibacterial Activity of the Protein Encoded by a Cryptic Open Reading Frame (cyt1Ca) from Bacillus thuringiensis subsp. israelensis by Site-Directed Mutagenesis

Mark Itsko,^* Robert Manasherob, and Arieh Zaritsky

Department of Life Sciences, Ben-Gurion University of the Negev, P.O. Box 653, Be'er-Sheva 84105, Israel

Received 13 February 2005/ Accepted 9 May 2005

Insecticidal crystal proteins of Bacillus thuringiensis belong to two unrelated toxin families: receptor-specific Cry toxins against insects and Cyt toxins that lyse a broad range of cells, including bacteria, via direct binding to phospholipids. A new cyt-like open reading frame (cyt1Ca) encoding a 60-kDa protein, has recently been discovered (C. Berry et al., Appl. Environ. Microbiol. 68:5082-5095, 2002). Cyt1Ca displays the structure of a two-domain fusion protein: the N-terminal moiety resembles the full-length Cyt toxins, and the C-terminal moiety is similar to the receptor-binding domains of several ricin-like toxins, such as Mtx1. Neither the larvicidal activity of cyt1Ca expressed in Escherichia coli nor the hemolytic effect of His-tagged purified Cyt1Ca has been observed (R. Manasherob et al., unpublished). This was attributed to five amino acid differences between the sequences of its N-terminal moiety and Cyt1Aa. The 3' end of cyt1Ca was truncated (removing the ricin-binding domain of Cyt1Ca), and six single bases were appropriately changed by site-directed mutagenesis, sequentially replacing the noncharged amino acids by charged ones, according to Cyt1Aa, to form several versions. Expression of these mutated cyt1Ca versions caused loss of the colony-forming ability of the corresponding E. coli cells to different extents compared with the original gene. In some mutants this antibacterial effect was associated by significant distortion of cell morphology and in others by generation of multiple inclusion bodies spread along the cell envelope. The described deleterious effects of mutated cyt1Ca versions against E. coli may reflect an evolutionary relationship between Cyt1Aa and Cyt1Ca.

Present address: Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305-5120.

This article has been cited by other articles:

• Manasherob, R., Itsko, M., Sela-Baranes, N., Ben-Dov, E., Berry, C., Cohen, S., Zaritsky, A. (2006). Cyt1Ca from Bacillus thuringiensis subsp. israelensis: production in Escherichia coli and comparison of its biological activities with those of other Cyt-like proteins.. Microbiology 152: 2651-2659 [Abstract] [Full Text]